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Objective To develop a rapid, sensitive and specific assay method, based on multiplex real time PCR for identifying Vibrio cholerae and distinguishing Vibrio cholerae O139 serotype from Vibrio cholerae. Methods Cholera toxin A subunit gene (ctxA) and glycosyltransferase gene (LPSgt) were chosen as targets according to Vibrio cholerae and Vibrio cholerae O139 serotype,and then the primers and TaqMan-MGB probe were designed. The 5'end of probes was labeled with FAM and VIC fluoresceins respectively while the 3' end of probes was labeled with MGB. The PCR reaction was optimized systematically. Then the specificity, sensitivity and reproducibility of multiplex real time PCR were estimated. Finally, multiplex real time PCR was applied to detect the clinical specimens. Results Vibrio cholerae was identified by multiplex real time PCR accurately and quickly,which could distinguish Vibrio cholerae O139 serotype from Vibrio cholerae. Vibrio cholerae was identified positive for primer pairs and probes from ctxA gene, and Vibrio cholerae O139 serotype tested was positive for LPSgt gene. Meanwhile, none of other bacteria was identified. Sensitivity of the test was 2 × 102 cfu/ml. When this assay was applied directly to identify 45 clinical specimens, results showed that 10 were positive to Vibrio cholerae, in which 4 clinical specimens were positive to Vibrio cholerae O139 serotype. All the results were the same to the one that had been obtained from the conventional assays. Conclusion Our rsults showed that the multiplex real time PCR was a reliable,accurate and feasible method for identifying Vibrio cholerae that carrying toxin gene and distinguishing Vibrio cholerae O139 serotype from Vibrio cholerae. This method could be applied to the cholera surveillance, prevention and control system for identifying and distinguishing Vibrio cholerae in the labs.
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<p><b>OBJECTIVE</b>To develop a loop-mediated isothermal amplification (LAMP) method for rapidly diagnosing of Escherichia coli (EHEC) O157:H7 in pathogen detection department or small-scale laboratories.</p><p><b>METHODS</b>Primers for LAMP test were designed by targeting the antigen coding rfbE of EHEC O157:H7, the Shiga-like toxin stx2 and the fliC encoding gene of H7 flagella antigen, respectively. The reaction condition and reaction system of LAMP were optimized. 2 EHEC O157:H7 type strains, 17 local strains and 33 other enterobacteria were analyzed to evaluate the LAMP's specificity and sensitivity. The results of the LAMP reaction were also compared with routine PCR method.</p><p><b>RESULTS</b>The amplification products of O157 which had the corresponding target genes turned green by visual inspection and had ladder-like pattern on the gel, but products of other enterobacteria remained orange by visual examination and had no band on the gel. The detection results of LAMP were the same as of routine PCR method. The reaction time of the LAMP method was only 1.5 hours and the detection limit of LAMP assay was 26 CFU/reaction. In addition, the LAMP results could be determined only by visual inspection.</p><p><b>CONCLUSION</b>LAMP assay is rapid, specific, and sensitive for the detection of EHEC O157:H7. This method might not only reduce the dependence of complicated equipments but also be a potential method for wider use in pathogen detection department, small-scale laboratory, emergency motor vehicle or field survey.</p>
Subject(s)
Environmental Monitoring , Methods , Escherichia coli O157 , Genetics , Nucleic Acid Amplification Techniques , Methods , Sensitivity and SpecificityABSTRACT
Objective To develop a loop-mediated isothermal amplification (LAMP) method for rapid diagnosing of Legionella pneumophila in the Pathogen Detection Department(PDD) or in small-scale laboratory. Methods Five primers (2 Inner Primers, 2 Outer Primers and a Loop Primer) for the LAMP test were designed by targeting the mip gene of Lpneumophila and reaction system of LAMP reaction was optimized. 12 strains of L.pneumophila, 45 local strains, 6 non-L.pneumophila strains, 11 other strains and 59 environmental water samples were analyzed to evaluate the specificity and sensibility of the LAMP amplification. At the same time, the results of the LAMP were also compared with biochemical culture and quantitative PCR methods. Results The amplification products of L.pneumophila turned green by visual inspection and had ladder-like pattern on the gel, but non-L.pneumophila and other products from the strains remained orange by visual examination and had no band on the gel. The detection rate of LAMP was higher than the biochemical culture and the real-time PCR methods. Reaction time of the LAMP method was only 1.5 h and the detection limit of LAMP assay was 5 cfu/reaction. In addition, the LAMP results could be determined only by visual inspection. Conclusion LAMP assay targeting the mip gene of L.pneumophila appeared to be rapid, specific, and sensitive for the detection of L.pneumophila. This method not only reduced the dependence of complicated equipment but also had a potential for wider use in the PDD, small-scale laboratory, emergency motor vehicle or for field survey.
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Objective To identify the presence of candidate pathogenicity island 89K DNA sequence of Streptococcus suis serotype 2 (SS2) strains isolated from patient in Zhejiang province. Methods Genes and DNA fragments were amplified by PCR, using specific primers, and three amplified fragments of the89K sequence were directly sequenced. The results were analyzed using software related to bioinformaties and epidemiology. Results 8 strains of SS2 all contained 89K sequence, cps2J and mrp virulent genes, and species-specific 16S rDNA. 3 amplified fragments of 89K candidate pathogenicity island of SS2 ZJ0501 were above 99% similar to SS2 strain identified from outbreaks in Jiangsu in 1998, and the gene fragment of coding DNA recombinant protein in the 89K sequence was highly homological with that of S. dysgalactiae and S. agalactiae. Conclusion In recent years SS2 strains isolaed from patients with clinical symptoms in Zhejiang province had been detected to have contained candidate pathogenicy 89K DNA fragment.